( Key words: ...)Multiplex protein detection with the ECL Plex fluorescent Western blotting system using the Ettan DIGE Imager

Key words: ECL Plex • Ettan DIGE Imager • fluorescent Western blotting • multiplex detection • dynamic range

The Ettan™ DIGE Imager is fully compatible with the ECL Plex™ system, reaching the same levels of sensitivity, linearity, and dynamic range as the Typhoon™ scanner. Similar results are also obtained with both scanners in multiplex applications, where two proteins can be detected in the same blot with minimal cross-reactivity between antibodies or dyes.

Introduction
The ECL Plex Western blotting system is based on the sensitive CyDye™ conjugated antibodies, enabling detection in the low picogram range with high linearity and a dynamic range of nearly four orders of magnitude (1). Multiplexing is another important property of the ECL Plex system, enabling blotting of two proteins simultaneously.

In an ECL Plex experiment, the data quality is not only dependent on the blotting, but also the imaging system used. A new scanning CCD camera, the Ettan DIGE Imager, is now available. It is a camera with high resolution specifically developed for 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) and the ECL Plex system, with the ability to produce multichannel images of Cy™2-, Cy3-, and Cy5 labeled gels and protein blots.

In this study we present a comparison of the Typhoon and the new Ettan DIGE Imager applied to the ECL Plex system. A number of CyDye conjugates were evaluated, using both the Hybond™ ECL (nitrocellulose) and the new low-fluorescent Hybond-LFP (PVDF) membranes in both single-protein and multiplex analyses. The blot scans were evaluated, resulting in data that was comparable between the two scanners, showing sensitive detection and a broad dynamic range. An application of the ECL Plex system with TGF-β–activated cells also resulted
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